INVESTIGATING THE EFFECT OF GINSENG … · Indian Journal of Fundamental and Applied Life Sciences ISSN: 2231– 6345 (Online) An Open Access, Online International Journal Available

Indian Journal of Fundamental and Applied Life Sciences ISSN: 2231– 6345 (Online)

An Open Access, Online International Journal Available at www.cibtech.org/sp.ed/jls/2015/02/jls.htm

2015 Vol. 5 (S2), pp. 1253-1264/Beigi et al.

Research Article

© Copyright 2014 | Centre for Info Bio Technology (CIBTech) 1253

INVESTIGATING THE EFFECT OF GINSENG TERATOGENIC ON THE

FETUS OF A FEMALE MICE BALB/C / C DURING

THE 7, 8,9,10 DAYS OF PREGNANCY

Soheila Azad Beigi1, *Parvin Torabzadeh

1 and Monire Movahedi

2

1Department of Biology (Cellular -Developmental Biology), Karaj Branch, Islamic Azad University,

Karaj, Iran 2Department of Biology, Tehran North Branch, Islamic Azad University, Tehran, Iran

*Author for Correspondence

ABSTRACT It is more than 5,000 years that the ginseng plant is being use in East Asian countries, especially Korea

and China as a healing agent to increase memory power, physical and sexual stamina, enhancing strength,

happiness and excitement and coping with aging Also this plant is used by athletes in the combination of

diet supplements to enhance their physical strength. For this reason, it was decided to study the eratogenic effects of this plant on adult female Balb / C during pregnancy. The objective of this research work was to

investigate teratogenic effects of ginseng plant on small mice embryos during pregnancy. Considering

that the Ginseng does not have lethal dose, Injection at a dose of 0/4 mg / kg intraperitoneally on days 7, 8,9,10 pregnancy after vaginal plug in adult female mice Balb / C with a weight of 28+_2 gr was done.

Along with the control group, the two experimental group and control group were considered to compare

answers. All rats were anesthetized by chloroform in day of 15 of pregnancy and the embryos were removed. The results of both the macroscopic and microscopic were analyzed. In microscopic study

abnormalities in embryos examined and the following results were obtained: Bleeding in more than 40%

of embryos in different areas of the body, anencephaly, asymmetric orientation of the limb, exohepatic,

defective growth of limb and C shape body. Conclusion: The ginseng plant has great medical and therapy properties most parts of the world use as a medicinal tonic for everyday applications but its use during

pregnancy is dangerous.

Keywords: Ginseng, Female Rat, Teratogenic, Pregnancy

INTRODUCTION Extensive research has been done about Ginseng in various fields but about its consumption during

pregnancy and its risks in this period and impact of this plant on embryo no research work has been done.

In this study it was attempt to investigate the effect of an aqueous solution prepared from 500 mg capsules

of this herb during pregnancy on fetal mouse of Balb / C. Iran due to its appropriate conditions to grow a variety of plants has adequate coverage of plants and exotic plants that among these plants that many of

them are known as a medicinal plant used in traditional medicine and people use them for treatment of

different disease in different ways such as boiled vegetables, edible, and dried. Ginseng is a native plant in China and Korea, which any species of that have not found in Iran yet.

About many properties of this plant and involved mechanisms and effective doses during pregnancy and

its effects on the fetus enough animal study have not done yet. The objective of this study was to

determine the teratogenic effects of this herb on mice embryos of Balb / C and comparing its complications at higher levels, such as a human. The reason to select this subject of study is due to lack of

national and international studies in this field. It is hoped that by doing this research to understand the

effects of this herb during pregnancy. Russian scientists have done more than 400 researches on healing effects of Siberian ginseng. The results of all investigations have shown that subjects who used brewed

ginseng are usually healthier, fresher, and in coping with adversity of live is endurance. A research group

affiliated with Tufts University of Washington in 2010 extracted quinonereductase from American ginseng which is effective in the prevention of premature aging in people (Chen and Ribaya, 2009).

In 2006, Japanese scientists extracted several substances from the root of this plant which are;




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Panksyn: increases the strength of muscles in the face of pain and pressure.

Panaks acid: modify the metabolism.

PANA Colin: effect on the endocrine glands. Panasn : stimulates the central nervous system because of having the Panoxid (Park et al., 2006).

In 2010, Japanese scientists have found that using oral ginseng to improve mood and anxiety in

postmenopausal women. This group believed that the main medical ingredients of ginseng are related to ginsinozid PPDS (Proto Panax Adiols) and PPTS (Proto Panax Atriols) that both dissolved in water well

and orally have a better effect (Xuet al., 2010).

A research group in 2013 with experiments on animal models has concluded that anti-fatigue effects of

ginseng are related to the impact of ganoids on the central nervous system (Kim and Park, 2013). In 2013 Karwah mentioned that red ginseng has a positive impact to treat erectile dysfunction in men (Leung and

Wong, 2013). Also in 2009, reported that ginsinoxide Rb2 improve the capacity of spermatogenesis in

human through Yeshn anti oxygen pathway (Choon and Junglimlee, 2009). Ebrahimi et al., (2011) in a research work in the treatment of cancer rats with busulfan in combination with ginseng and after

preceding the treatment phases realized that taking ginseng during treatment with the drug, improve

spermatogenesis. So that shows increase in stem cells of spermatogonial and spermatozoa. In this study it was observed that ginseng as a plant contains phytoestrogens gincinoxid, the disorders of busulfanas a

chemotherapy drug with alkylating properties in germinal epithelium and reduces semen parameters.

Pak and Lim (2005) have studied the effect of red ginseng on polycystic ovary syndrome in rats and

concluded that ginseng increases induced growth factor (NGF) in the ovaries (Sawiress and et al., 2011). Studied the effect of ginseng on testicular function in rats with diabetes and concluded that inducing

ginseng diabetic rats prevents the degenerations of somniferous tubules.

MATERIALS AND METHODS

In order to prepare Ginseng solution and study its teratogenic effects on the fetus mice of Balb / C was

performed according to the following steps:

Ginseng capsules were purchased from pharmacies. To prepare ginseng solution for Intra Peritoneal injection, 500 mg of each capsule distilled in 10ml of distilled water is poured into the flask and solved.

Because ginseng hardly dissolves in water, put the prepared suspension in a shaker for 2 hours until well

dissolved. Then the solution is passed through a filter, pour the similar amount of solution into the Falcon tubes and centrifuges at 4000 rpm for 15 minutes. After the centrifugation, the solution is ready for

injection. Laboratory rats were purchased from the Institute of Razi Vaccine and Serum Research Institute

of Hisarak. Then they were kept in animal room with controlled conditions of light, temperature and humidity. Using the automatic timer, light frequency of 12 hours light and 12 hours of darkness was

restored. Room temperature using heaters and heating during the winter and cooler in summer was

adjusted at 21+_2 °C at normal humidity (RH 50-40%). Rat cages each three days cleaned and washed

and once a month it was autoclaved and fine wood chips were cast. Their water glasses were controlled and cleaned daily. Prepared rat food purchased from Livestock and Poultry Company and is controlled on

a daily basis. In order to determine the specific days of pregnancy, adult female and male rats 2.5-3

month; weighing 24- 28 g were placed in special cages for intercourse with polygamy method. Through observing Vaginal Plug the zero day of pregnancy determined. Intercourse was between 10 pm to 1 am.

Therefore, the bases of starting pregnancy considered at 12 midnight. In this way, for injection during

determined days of pregnancy after observing vaginal plug, separate male and female rats and were kept in individual cages.

To study the biological effects, prepared aqueous solution of plant and injecting it to the pregnant female

rats, determine the LD50 dose at first and multiple doses of 24 to 26 mg injected into rats but there was

no mortality in any of the rats. Then, according to the rat weigh the amount of 0.4 g/kg is considered for injection. Before injection, the rats were separated into 4 separate groups of 10.

Group 1 (control) consisted of 10 animals that did not receive any drug or solvent during the experiment.

Group 2 (sham): consisted of 10 animals that distilled water were injected




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Group 3 (experiment 1) considered of 10 animals that consecutively in day of 7 of pregnancy 0.4g/kg

aqueous solution was injected.

Group 4 (experiment 2) included 10 animals that consecutively was injected 0.4g/kg aqueous solution in 8

th day of pregnancy

Group 5 (experiment 3) of 10 animals that consecutively was injected 0.4g/kg aqueous solution in 9th day

of pregnancy Group 6 (experiment 4) was of 10 animals that consecutively was injected 0.4g/kg aqueous solution in

10th day of pregnancy

All the rats were killedby chloroform in 15th day of pregnancy and dissectioned.

Injection Method Prepared solution was injected Intra Peritonial to pregnant female rats. The reason for this choice was fast

absorption of injected substances and their entry into the circulatory system. Before the injection, the rats

were weighed carefully. A total of 60 adult female rats put into 6 groups of 10. After we recognize that Ginseng doesn’t have LD50 with 0.4 dose applied the injection on experimental groups. All injections

were performed at 10-11 am.

Exiting the embryos and fixation process: After the injection in 15

th day of pregnancy, dissection was performed. The rats were killed by chloroform

and immediately after death, dissection embryos into the uterus with the help of special equipment exited

and placed in normal saline. Then amniotic sac surrounding the fetus was removed carefully.

After washing the embryos in physiological saline solution, with help of caliper the length of Crown-Rump (CR) measured and through precise balance the weight of embryos and placentas determined and

recorded. After initial morphological studies, embryos and pairs of experimental and control group were

fixed in 10% formalin fixative solution for 24-18 hours.

Methods of Fetal Tissue Sections

Prepared fetal samples were stored in formalin. Then for sectioning and staining was referred to the TMU

laboratory.

The fourth step is the preparation of slides: Slides were placed into the oven for 10 minutes until paraffin melted completely.

Samples were fixed on slides.

Histological and cytological methods of studying sample: For more accurate study, the Effects of ginseng solution on embryos and control, prepared slides

examined carefully with stereo photomicroscope and microscope.

In macroscopic study some abnormalities were observed in embryo. The desired sections were photographed.

Applied Statistical Methods

Since in all the experiments, experimental samples were compared with control and sham groups.For

statistical analysis of the results, One Way ANOVA with repeat was used. Tables were prepared. SPSS software was used for data analysis. Statistical analysis of data obtained from the operation was carried

out with the help of computers. Desired parameters including mean, standard deviation and variance

analysis. In this series of experiments dosage of 0.4 g/kg aqueous solution of Ginseng were injected Intra Peritoneal to pregnant rats in days of 7, 8,9,10 with hygiene. To compare experimental samples and

controls with 3 times repeating which means 15 pregnant female rats were considered as control and

witness. So that a series of rats without any injection considered as a control and the other series (witness group) were injected with distilled water. Experimental, control and witness rats were dissection in the

15th day of pregnancy. Macroscopic examination revealed that all embryos of control and witness group

were healthy. However, the experimental rats that aqueous solution of plant in days 7 to 10 of pregnancy

was injected, fetuses with abnormalities was obtained. The results of injecting ginseng in days of 7, 8, 9, and 10 of pregnancy

The results of injecting ginseng in 7th day of pregnancy




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30 of experimental embryo at 7th day of injection 10 fetuses were apparently healthy (33.4%). A total of

20 embryos had various anomalies (66.6%) which include 7 fetal that showed exohepaticab normality

(23.3%) 1 fetus showed exence phalus (3.03%). 5 embryos showed bleeding under the skin(16.6%) and 5 fetus had umbilical hernia(16.6%) and 2

embryos showed a C-shaped body(6.06%) and 4 rats had uterus with atrophicfetus (40%) and 2 rats had

thicken uterus (20%).

The results of Ginseng Injection on Day 7th

of Pregnancy

According to the table, it can be said that injecting Ginseng Intra Peritonialy in the 7th days of pregnancy

causesfetal weight, placental weight, CR embryo and placenta diameter decreases significantly with p

value <0.001. It is clear in 1-3 to 3-4 histograms. In histograms of 1 to 4 it is completely clear.

Histogram 1: Comparing the weight of experimental embryos, sham and control in the 15

th day of

pregnancy of the 7th

day of pregnancy experiment series (gr) (P <0.001))

Histogram 2: Comparing the weight of experimental embryos, sham and control in the 15

th day of


day of pregnancy experiment series (gr) (P <0.001))




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Histogram 3: Comparing the size of CR in experimental, sham and controlembryos in the 15

th day

of pregnancy of the 7th

day of pregnancy experiment series (mm) (P <0.001)

Histogram 4: Comparison of the experimental pairs in diameter, sham and control in the 15

th day



The Results of Ginseng Injection on 8th

Day of Pregnancy In this series of experiments dosage 0.4 mg/kg.bw was injected to the pregnant rats on 8

th day of

pregnancy. For comparative analysis, experimental samples with the same number of controls, the control

rats considered as sham. So that control rats didn’t injected and distilled water was injected sham group.

Embryos of experimental, control and sham were isolated on the 15th day of pregnancy from the mother

womb. Initial macroscopic examination revealed that all embryos of sham and control were healthy. A

total of 38 experimental embryos on the 8th day of injection 7 embryos were apparently healthy(18.43%),

31 fetuses had various abnormalities (81.57%) that include; 6 fetuses had exohepatic abnormalities (15.78%) 1 embryo had Exencephalus (2.63%), 1 fetus had a C-shaped body(2.63%) . 8 fetuses showed

bleeding under the skin(21.02%) and 6 embryos had inguinal hernias (15.78%) and 6 rats had thickened

uterus and atrophic embryo (60%).


Day of Pregnancy




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According to the table, we can say that injecting ginseng Intra Peritonially on the 8th day of pregnancy

causes fetal weight, placental weight, embryo CR fetal placental diameter significantly decrease with p

value <0/001. In histogram of 5 to 8 it is clear.

Histogram 5: Comparing the experimental fetus weight, sham and control on the 15

th day of


day of pregnancy experiment series (gr) (P <0.001)

Histogram 6: Comparing the weight of experimental pairs, sham and control on the 15

th day of


day of pregnancy experiment series (gr) (P <0.001)

Histogram 7: Comparing the size of CR in experimental, sham and control embryos on the 15

th day






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Histogram 8: Comparing diameters of experimental, sham and control embryos on the 15

th day of



The Results of Injecting Ginseng on 9th

Day of Pregnancy In this series of experiments dosage of 0.4mg/kg.bw were injected intraperitoneally to pregnant rats on 9

th

day of pregnancy. In order to comparative analysis experimental samples with the same number of

controls were considered as sham in rats. So that the control rats didn’t inject and the distilled water was injected to sham group. Experimental, control and sham were isolated from the mother womb on the 15

th

day of pregnancy. Initial macroscopic examination revealed that all embryos of control and sham were

healthy. A total of 35 experimental embryos of 9th day of injection, 7fetuseswere apparently healthy

(20%), 28 of foetuses had various anomalies (80%) that are;

7 of embryos showed exohepatic abnormality (20%) 3 foetus showed exencephalus (8.57%). 2 fetuses

had a C-shaped body (5.71%).8 fetuses showed bleeding under the skin (22.58%) and 6 embryos had

inguinal hernias (17.14%) and 2 fetuses had asymmetric navigation of foot and hand and lack of foot formation (5.71%) and 1 rat with thickened uterine and 5 rats had uterine with atrophic fetus that they

were 6 infertile uterine (60%).


Day of Pregnancy According to table (6-3) it can be said that injecting ginseng intraperitoneally on 9

th day of pregnancy

cause decrease significantly in the Fetal weight, Placental weight, fetus CR, and placenta diameter with p

value <0/001. It is obvious in histograms of 9 to 12.

Histogram 9: Comparing the weight of experimental, Sham and control embryo, on the 15

th day of

pregnancy of experiments of 9th

day of pregnancy (gr) (p value <0/001)




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th day of



Histogram 11: Comparing the size of CR in experimental, Sham and control embryo on the 15

th day

of pregnancy of experiments of 9th

day of pregnancy (mm) (p value <0/001)

Histogram 12: Comparing diameters of experimental, Sham and control pairs on the 15

th day of




Day of Pregnancy In this series of experiments dosage of 0.4mg / kg.bw soluble ginseng, intraperitoneally into pregnant rat

were injected on day 10 of pregnancy. Comparative analysis of experimental samples with the control,

with the same numbers control rats considered as sham. So that nothing were injected to control rats and distilled water was injected into sham. Experimental, control and sham embryos were isolated on the 15

th

day of pregnancy from the mother womb. Initial macroscopic examination revealed that all embryos of

control and sham were healthy. 17 of experimental embryo on the 10th day of injection, and 2 fetus were




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apparently healthy (11.76%) 15 of the embryos had various abnormalities (88.24%) that include; 3 fetus

showed exohepaticabnormalities (17.64%), 3 fetuses were with a small placenta (17.64%), 5 fetus showed

bleeding around the ears and spine (29.41%) 3 fetuses were withumbilical hernia (17.64%) and also 1 fetus showed tilt of the head from the axis of body (5.88%) and 5 fetus were with thickened uterus and

had atrophic embryo (50%).

Examine the Results of Ginseng Injection on 10th

Day of Pregnancy Injecting ginseng intraperitoneally on the 10

th day of pregnancy causes significant decrease in Fetal

weight, fatal CR, and placenta diameter with p<0/001 (Histopathology grams, 13, 15, 16). Placental

weight decreased but it was not significant (histogram 14).


th day of




th day of


day of pregnancy (gr)




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Histogram 15: Comparing the size of CR in experimental, Sham and control embryo on the 15

th day

of pregnancy of experiments of 10th


Histogram 16: Comparing the diameter of pairs of experimental, Sham and control on 15

th day of

pregnancyof experiments of 10th


The Results of Studies on Fetuses with Exohepatic Abnormalities (Herniated Liver)

Macroscopic and microscopic examinations of fetuses with exo hepatic abnormalities in comparison with control embryos reveals that liver enlarged, abdominal wall from the chest to the abdomen and flank area

is opened and theabdominal wall from the chest to the abdomen and flank area is opened and the liver is

seen out of the body. This abnormality observed in each 4 days of pregnancy. Of course with different

percentages, on the 7th

day of pregnancy (23.3%) which is the highest percentage in the injection of 7th

day. 8

th day of pregnancy (15.78%), 20

th day of pregnancy (20%) and 10

th day of pregnancy (17.64%) was

observed.

Examine the Results of Studies on Fetal with Abnormalities in the Limb It was observed in macroscopic examination that there is asymmetric orientation in limbs of hand, foot

and also the feet are not formed in experimental embryos which were observed on 9th day of pregnancy.

Examine the Results of Studies on Embryos with Subcutaneous Hemorrhage

It was observed in the macroscopic examination, that there are subcutaneous hemorrhages in different parts of the body including the scalp, face, and ears, lumbar and so on. Sometimes there is bleeding in the

leg and tail. The bleeding was observed on different days with different percentages so that on the 7th day




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of pregnancy (16.6%), on the 8th day of pregnancy (21.02%), on the 9

th day of pregnancy( 22.85%), and

on the 10th day of pregnancy( 29.41%) that these abnormalities were more than the other days in the

injection of 10th day of pregnancy.

Examine the results of studies on embryos with xencephalus (protrusion of the brain):

Examine the embryos with exencephalus through stereomicroscope revealed the brain protrusions. For

more detailed analysis, Microscopic sections were prepared from the exencephalusembryos that the abnormality observed on different days of gestation with different percentages so that on the 7

th day of

pregnancy (3.03%), on the 8th

day of pregnancy( 2.63%) and on the 9th day of pregnancy (8.57%) was

observed. This abnormality was not observed on the 10th day of pregnancy. The highest percentage of this

abnormality was on the 9th day injection.

Examine the performed studies on fetuses with umbilical hernia:

In the macroscopic and microscopic examination experimental embryos, had a hiatal hernia so that

different percentages observed on different days. On the 7th day of pregnancy (16.66%), 8

th day of

pregnancy (15.78%), 9th day of pregnancy (17.14%) and on the 10

th day (71.64%) was observed.

Examine the performed studies on fetuses with C- Shape body:

In macroscopic and microscopic examination, some of the experimental embryos had c- shape body so that different percentages observed on different days. On the 7

th day of pregnancy (6.06%), on the 8

th day

of pregnancy (2.63%), on the 9th

day of pregnancy (5.71%) and on the 10th day of pregnancy this

abnormality did not observe.

RESULTS AND DISSCUSSION

Ginseng solution was injected intra peritoneally into adult rats. In this method the substance is rapidly

absorbed into the visceral peritoneum and then entered to the liver and bloodstream. In the present research several abnormalities observed due injecting ginseng on 7, 8, 9,10days of pregnancy which

include;

In this series of experiments, injecting ginseng with dosage of 0.4 mg / kg.bw causes abnormal embryos

in the nervous system with Exencephalus, so that abnormities observed as follow; on the 7th day of

pregnancy (3.03%), on the 8th

day (2.63%), on the 9th day (8.57%). Probably meningeal membrane is not

formed in different parts of the brain and protrusion of the brain has occurred (5).

According these results, ginseng can be introduced as a neurotropic teratogen. Compare the size of the crest- rump (CR) in 15 days experimental embryos revealed a decrease in

comparison with other groups of control and sham so that significant decrease in embryos CR observed

on days of 7,8 and 9 of pregnancy (p<0.001) and on 10th day of pregnancy(p<0.05). Probably, ginseng had inhibitory effect on the growth hormone of the pituitary gland. The pituitary gland

has a buff form structure and located at the basis of brain. This gland is called the leader. The pituitary

gland secretes several hormones.

The hormone that plays an important role in growth of organ is Somatropin or growth hormone. This hormone has an important role in growth and bone formation (height, arm and leg length) (4) that with

reduction of height, weight and organs in experimental rats we conclude that ginseng has a significant

impact on growth hormone and causes the CR reduction. Ina series of experiments of injecting ginseng with dosage of 0.4 mg/kg.bw on days of 7, 8, 9 and 10 the exohepatic abnormality observed, so that on

the 7th day of pregnancy (23.3%), on the 8th day (15.78%), on the 9th day of pregnancy (20%) and on the

10th day of pregnancy was (17.64%) due to the non formation of the peritoneal membrane from the chest to the end of abdominal area caused the herniated liver.

Also it was observed that abnormalities in limb or non formation of limb or Incomplete formation of

organs, so that on the 9th day (5.71%) this abnormality were observed that according to these results

ginseng can be introduced as a kind of teratogene for the growth and development of limb. Ginseng directly effects on the fetus during organogenesis (rat embryo). Limb development begins when

themesenchymal cells of the partial layer or somatic mesoderm of lateral plate and xylotomy mesoderm

(part of the spine) with the each other accumulate under the ectodermic.




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Tissue and creates a spike named limb bud. Most of the limb bud cells after performing its function

undergo apoptosis and only the expressing cells of BMP remains. These cells preferentially convert to

bone, muscle and the skin of the posterior limb. In fact the posterior limb of rats is created by the BMP-secreting cells. So we can mention that ginseng make damage to the limb bud and the genes that should

be expressed on that part for the formation of limbs and fingers and inhibit expression of genes and causes

the creation of fetus with limb abnormality (not forming organs, defective formation of organs, not the formation of the fingers or toes) (13). When there is a defect in the expression of Chordinand Noggin

genes in rat, the formation of eye, ear and limb faces problem (3) probably ginseng affect the expression

of these genes and their expression faces the problem. In macroscopic studies, some experimental fetuses

after injecting ginseng with dosage of 0.4 mg/kg had severe bleeding under the skin on the scalp, face, ears, body, and tail so that on the 7th day of pregnancy(16.6%), on the 8th day of pregnancy( 21.02%), on

the 9th day of pregnancy( 22.85%) and on the 10th day of pregnancy(29.41%) of this abnormality was

observed which is probably due to the non formation of the Platelets or defective formation of them sever subcutaneous bleeding occurred. Platelets are the spherical or elliptical form

Bodies which are produced from the segmentation of large cells cytoplasm in the bone marrow that

called megakaryocytic. Mega karyocytes are the stem cells of the platelets and their non- differentiation cause decrease of platelet and consequently severe bleeding under the skin can occur. Platelets are non-

core. Platelets are not only through the contraction of vascular smooth blood clotting but also help

prevention of bleeding(13) Studies show that injection of Ginseng reduces blood platelets and results in

severe bleeding under the skin and placenta.

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Documents

INVESTIGATING THE EFFECT OF GINSENG … · Indian Journal of Fundamental and Applied Life Sciences ISSN: 2231– 6345 (Online) An Open Access, Online International Journal Available